Addiction of anti-CD28 antibodies restores PBMC proliferation and IFN-gamma production in lepromatous leprosy patients.

During antigen recognition, T lymphocytes are primed by a physical interaction with antigen-presenting cells (APC). At least two signals are needed to activate T cells. One is provided by T cell receptor (TCR)/CD3 in the context of the mayor histocompatibility complex (MHC), and another signal is mediated by antigen-independent molecules, that is T cell membrane-bound CD28 and its specific ligand B7-1 (CD80) present in APC. Both signals trigger a series of metabolic events initiating right at the cell membrane and ending with activation and proliferation of T cells as well as specific cytokines synthesis. Our main goal was to determine whether deficiency in interferon-gamma (IFN-gamma) production shown by peripheral blood mononuclear cells (PBMC) from lepromatous leprosy (LL) patients, could be overcome by reconstituting in vitro the appropriate signals (by means of addition of anti-CD28 and anti-CD80 monoclonal antibodies). We also determined the stimulation index (SI) in the same PBMC. Our results demonstrated no significant differences in CD80 expression monocytes and B lymphocytes from LL patients when compared with healthy subjects. Nonetheless, CD28 expression significantly decreased in lymphocytes from LL patients (p < 0.01). Regarding IFN-gamma levels and SI, LL-PBMC failure before mitogenic stimuli could be reversed by further incubation with anti-CD28 antibody, but stimulation by specific antigen of Mycobacterium leprae was not changed. Addition of anti-CD80 antibody significantly increased IFN-gamma levels in phytohemagglutinin (PHA)-stimulated PBMC, although proliferation deficiency persisted. Cells stimulated with specific antigen did not modify either their proliferation or IFN-gamma levels.

Effect of phorbol myristate acetate (PMA) and ionophore A23187 on interleukin-2 levels and proliferation of activated T lymphocytes from patients with lepromatous leprosy.

The immunodeficiency present in patients with lepromatous leprosy is characterized by the limited proliferation of T lymphocytes, and is explained in part by the impaired synthesis of interleukin-2 (IL-2). Diacylglycerol (DAG) and calcium produce the activation of PKC, ERK and JNK kinases, implying a normal IL-2 response. Phorbol esters, such as PMA, can substitute for DAG and are mitogenic to human T and B cells activating several cytokine-encoding genes. Ionophore A23187 increases calcium permeability across the cellular membrane to the cytosol of lymphoid cells and is considered a co-mitogen of T lymphocytes. Here we report that: 1) PHA-activated T lymphocytes from LL patients can be separated in vitro into two groups: a) responders (R) with a stimulation index (SI) of > 10 and (b) nonresponders (NR) with a SI of < 10. 2) The proliferative responses of cells from LL(R), LL(NR) and normal subjects were measured after being stimulated with: I, PHA, PMA, PMA + I PHA + PMA and PHA + PMA + ionophore (PPI). The most important result occurs in LL(NR) patients whose cells did not respond to PHA stimulation but increased to normal levels of proliferation when they were stimulated with PMA. Furthermore, the three groups, (NR, R and normals) strongly increased their responses when they were incubated with PPi. 3) Finally, Il-2 concentrations in the supernatants of cultures of T lymphocytes from LL(NR), LL(R) and controls were relatively low when they were incubated with PHA or PMA, but the addition of ionophore to PMA and the combination of PHA + PMA strongly increased the production of IL-2 in all of them, reaching the optimum IL-2 concentration when PPI is used. It can be concluded that the use of PMA, analogous to DAG, and ionophore A23187 (calcium increaser) in cultures of mitogen-activated T lymphocytes from LL patients induced the expression of the IL-2 gene, thus correcting the inadequate proliferation of T cells from LL patients.

Recognition of Mycobacterium leprae antigens with antibodies present in sera from patients with lepromatous leprosy.

A great diversity of antigens from Mycobacterium leprae have been described. One practical approach should be to utilize them as markers to indicate when a household contact is at risk of becoming infected and then moving to an active form of leprosy. For this purpose, sonic extracts of M. leprae were fractionated in 10% SDS-PAGE under reducing conditions. The fractionated proteins were then transferred to nitrocellulose sheets and incubated with sera from lepromatous leprosy cases, their contacts, and normal subjects in order to reveal the frequency of antigen recognition of each set of sera. The results showed that sera from lepromatous leprosy patients frequently recognized two proteins, one of approximately 28 kDa and the other of approximately 65 kDa, when compared with the sera from normal subjects. The contacts frequently recognized an approximately 16-kDa antigenic band, while sera from normal subjects recognized one protein of approximately 18 kDa. According to the results, the four recognized proteins from M. leprae can be considered markers of the above conditions (approximately 65 kDa, approximately 28 kDa for lepromatous leprosy, approximately 16 kDa for contacts, and approximately 19 kDa for normal subjects). From these, an easy serological test, such as an ELISA, can be developed to predict if a contact is moving toward lepromatous leprosy before detection of the actual clinical signs or symptoms.